Fig. 2. Oxa1p interacts with proteins encoded by mitochondrial DNA. In vitro translation was performed for 15 min at 25°C in wild-type mitochondria in the presence of [³⁵S]methionine, then the chemical cross-linker SPDP (0.5 mM) (A) or DSP (0.2 mM) (B) was added. The control samples (–) were mock-treated in parallel and received the solvent, dimethyl sulfoxide (DMSO), alone. Translation was allowed to proceed for another 15 min at 25°C. Cross-linking and translation were stopped by the addition of 100 mM glycine and 20 mM unlabeled methionine pH 8.0. Mitochondria were re-isolated, washed and lysed. After a clarifying spin, solubilized cross-linked products were immunoprecipitated with either pre-immune serum (PI) or antiserum specific for Oxa1p (α-Oxa1). Precipitated cross-linking products were eluted from the PAS-beads with sample buffer containing β-mercaptoethanol, which cleaves the cross-linker. Translation products were analyzed by SDS–PAGE and autoradiography. Total, 2% (A) or 1% (B) of cross-linking reaction that was used for immunoprecipitation. Note, in (B) less Oxa1p and pre-immune antisera was used for the immunoprecipitation reaction; thus, a reduction in the Var1p background signal was observed in this experiment.